Influence of flavonols and quercetin derivative compounds on MA-10 Leydig cells steroidogenic genes expressions

Article abstract of DOI:10.1016/j.tiv.2017.06.027

Androgen are mainly synthesized and secreted from testicular Leydig cells and play critical roles in testis development, normal masculinization, spermatogenesis, and male fertility. The rate-limiting step in testosterone biosynthesis involves the import of cholesterol inside mitochondria by the steroidogenic acute regulatory (Star) protein. Cholesterol is then converted to pregnenolone by the steroidogenic enzyme Cyp11a1, followed by a chemical transformation to testosterone using other steroidogenic enzymes. Interestingly, levels of Star protein within adult Leydig cells decrease during aging, resulting in defective mitochondrial cholesterol transfer and reduced testosterone production. Such decline may be delayed by increasing Star and/or Cyp11a1 gene expressions using supplementation with flavonoids, a group of the polyphenolic compounds widely distributed in fruits and vegetables. In this study, we examined whether the distribution of hydroxyl groups and/or acetylation or methylation of flavonols could influence their potency to stimulate steroidogenesis within Leydig cells. Low levels of quercetin, myricetin and pentaacetylquercetin (10 μM) stimulated cAMP-dependent Star, Cyp11a1 and Fdx1 promoters' activations and may increase steroidogenesis within Leydig cells. Indeed, pentaacetylquercetin successfully increased cAMP-dependent accumulation of progesterone from MA-10 Leydig cells, possibly through activation of Star and Cyp11a1 transcriptions. Thus, dietary supplementation of pentaacetylquercetin could be potentially effective to maintain testosterone production within aging males.

Full text of DOI:10.1016/j.tiv.2017.06.027

Accepted Manuscript
Influence of flavonols and quercetin derivative compounds on
MA-10 Leydig cells steroidogenic genes expressions
Michelle Cormier, Firas Ghouili, Pauline Roumaud, Luc J.
Martin, Mohamed Touaibia
PII:
DOI:
Reference:
S0887-2333(17)30183-2
doi: 10.1016/j.tiv.2017.06.027
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Toxicology in Vitro
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11 June 2017
26 June 2017
Please cite this article as: Michelle Cormier, Firas Ghouili, Pauline Roumaud, Luc J.
Martin, Mohamed Touaibia , Influence of flavonols and quercetin derivative compounds
on MA-10 Leydig cells steroidogenic genes expressions, Toxicology in Vitro (2017), doi:
10.1016/j.tiv.2017.06.027
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Influence of flavonols and quercetin derivative compounds on
MA-10 Leydig cells steroidogenic genes expressions
Running Title: Polyphenolics on MA-10 Leydig cells
Michelle Cormier1, Firas Ghouili1, Pauline Roumaud1, Luc J. Martin1*, Mohamed
Touaibia2
1
Biology Department, Université de Moncton, Moncton, New-Brunswick,
Canada, E1A 3E9.
2
Chemistry and Biochemistry Department, Université de Moncton,
Moncton, New-Brunswick, Canada, E1A 3E9.
*Corresponding author:
Dr. Luc J. Martin
Biology Department
Université de Moncton
18, avenue Antonine Maillet
Moncton, New-Brunswick
CANADA E1A 3E9
Tel: 506-858-4937
Fax: 506-858-4541
E-mail: Luc.Martin@umoncton.ca

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Abstract
Androgen are mainly synthesized and secreted from testicular Leydig cells and play
critical roles in testis development, normal masculinization, spermatogenesis, and male
fertility. The rate-limiting step in testosterone biosynthesis involves the import of
cholesterol inside mitochondria by the steroidogenic acute regulatory (Star) protein.
Cholesterol is then converted to pregnenolone by the steroidogenic enzyme Cyp11a1,
followed by a chemical transformation to testosterone using other steroidogenic
enzymes. Interestingly, levels of Star protein within adult Leydig cells decrease during
aging, resulting in defective mitochondrial cholesterol transfer and reduced
testosterone production. Such decline may be delayed by increasing Star and/or
Cyp11a1 gene expressions using supplementation with flavonoids, a group of the
polyphenolic compounds widely distributed in fruits and vegetables. In this study, we
examined whether the distribution of hydroxyl groups and/or acetylation or
methylation of flavonols could influence their potency to stimulate steroidogenesis
within Leydig cells. Low levels of quercetin, myricetin and pentaacetylquercetin (10 µM)
stimulated cAMP-dependent Star, Cyp11a1 and Fdx1 promoters’ activations and may
increase steroidogenesis within Leydig cells. Indeed, pentaacetylquercetin successfully
increased cAMP-dependent accumulation of progesterone from MA-10 Leydig cells,
possibly through activation of Star and Cyp11a1 transcriptions. Thus, dietary
supplementation of pentaacetylquercetin could be potentially effective to maintain
testosterone production within aging males.
Keywords: Quercetin / Myricetin / Retusin / Acetylquercetin/ Methylquercetin /
Steroidogenesis / Leydig

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Introduction
Testicular Leydig cells are major contributors of androgen synthesis and secretion,
playing an important role in testis development, normal masculinization, maintenance
of spermatogenesis, and male fertility. Indeed, Leydig cells dysfunctions may lead to
impaired male reproductive function, including primary hypogonadism, cryptorchidism,
and hypospadias. In addition, lower testosterone plasma levels may contribute to the
development of abdominal obesity in men (Cohen, 2001; De Maddalena et al., 2012;
Mårin and Arver, 1998) by reducing lipolysis of visceral fat (Mårin, 1995; Xu et al., 1990).
Such obesity condition is also responsible, in part, for reduced testosterone production
according to aging (Derby et al., 2006; Tan and Pu, 2002).
In Leydig cells, the critical rate-limiting step for testosterone biosynthesis involves
import of cholesterol inside the mitochondria by the inner membrane-bound
steroidogenic acute regulatory (Star) protein (Lin et al., 1995; Stocco and Clark, 1996;
Wang et al., 1998). Within mitochondria, cholesterol is metabolized by the steroidogenic
enzyme Cyp11a1 to pregnenolone, which is further converted to testosterone by other
steroidogenic enzymes of the endoplasmic reticulum. Interestingly, aging is associated
with reduced Star protein levels within adult Leydig cells, resulting in defective
mitochondrial cholesterol import and lower testosterone production (Culty et al., 2002;
Leers-Sucheta et al., 1999). As previously reported (Wang et al., 2005), age-related
decline in testosterone production may be delayed by increasing Star and/or Cyp11a1
gene expressions using supplementation with flavonoids or chemical derivatives.
Flavonoids are polyphenolic compounds widely distributed in fruits and vegetables.
These compounds have been reported to have anti-inflammatory, anti-cancer, and anti-
oxidative properties (Cárdenas et al., 2006; Ferrándiz and Alcaraz, 1991). In addition,
flavones, such as apigenin and chrysin, have been shown to increase androgen
production from testicular Leydig cells by increasing Star gene expression (Jana et al.,
2008; Li et al., 2011). Among common flavonol compounds, quercetin and myricetin
have been discovered in many dietary sources. Indeed, quercetin can be found in

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capers, peppers, onions, cranberries, apples, and tea (D’Andrea, 2015), whereas
myricetin is found in berries, tea, grapes as well as red wine (Somerset and Johannot,
2008). Quercetin has numerous pharmacological activities, including anti-oxidant, anti-
cancer, antiviral, anti-ischemic, anti-inflammatory and anti-allergenic actions. In this
study, we examined whether the addition of an extra hydroxyl as in myricetin,
acetylation or methylation of the hydroxyl groups (Figure 1) could influence their
potency to stimulate steroidogenesis within Leydig cells. The acetylation of quercetin
has resulted in greater bioavailability even if the ester groups are hydrolysable (Picq et
al., 1982). It will be interesting to compare the penta-acetylated analog with the penta-
methylated derivative which must be resistant to hydrolysis. The trimethyl analog can
also be interesting as the free hydroxyl increases the polarity of the molecule relative to
pentamethyl analog while allowing the molecule to have hydrogen bonds.
Thus, the current research aims to determine which flavonol derivatives have the most
potent stimulatory effect on testicular Leydig cells function by increasing steroidogenic
genes expression. Among them, quercetin, myricetin, pentaacetylquercetin, retusin and
penta-O-methylquercitin will be evaluated for their potential to activate Star, Cyp11a1
and Fdx1 promoter reporter constructs and to increase cAMP-stimulated progesterone
accumulation from MA-10 Leydig cells.

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Material and methods
Chemicals
Quercetin, myricetin and forskolin (FSK), were purchased from Sigma-Aldrich Canada
(Oakville, Canada). Purification of chemicals was carried out by flash chromatography
(CombiFlash®, Separation System SG 100C, ISCO). NMR spectra were recorded on a
Bruker Avance III 400 MHz spectrometer.
Synthesis of quercetin derivatives
Synthesis of pentaacetylquercetin
Pentaacetylquercetin
(2-(3,4-diacetoxyphenyl)-4-oxo-4H-chromene-3,5,7-triyl
triacetate) was synthesized, according to a published procedure (Picq et al., 1982), in
63% yield from quercetin and acetic anhydride as a yellow solid (Rf = 0.35
o
1
(EtOAc/hexane 50:50, mp = 195-198 C). H NMR (400 MHz, DMSOd6, 25°C) δ (ppm):
2.32 (s, 3H, 3-OAc), 2.33-2.34 (s, 12H, aromatic-OAc), 7.17 (d, 1H, H-6, J = 2.2 Hz), 7.53
(d, 1H, H-5’, J = 8.6 Hz), 7.64 (d, 1H, H-8, J = 2.2 Hz), 7.35-7.65 (m, 2H, H-2’, H-6’). 13C
NMR (100 MHz, DMSOd6, 25°C) δ (ppm): 169.79, 169.25, 168.70, 168.52, 168.26,
156.83, 154.86, 153.84, 149.96, 144.92, 142.65, 133.68, 127.56, 127.16, 124.99, 124.20,
115.19, 114.54, 110.52, 21.33, 21.22, 20.85, 20.81, 20.65.
Synthesis of retusin and Penta-O-methylquercetin
Retusin, ((2-(3,4-dimethoxyphenyl)-5-hydroxy-3,7-dimethoxy-4H-chromen-4-one), and
penta-O-methylquercetin were synthesized from quercetin, potassium carbonate, and
iodomethane. Even with an excess of 10 equivalents iodomethane, only retusin was
obtained after purification by flash chromatography (EtOAc/hexane: 0-80%) as a yellow
o
solid (yield: 48%, Rf = 0.63 (EtOAc/hexane 50:50), mp = 160-162 C). The obtained
1
1
product has the same H NMR as described in the literature (Moalin et al., 2011). H-
NMR (400 MHz, CDCl3, 25°C) δ (ppm): 3.88 (s, 3H, 3′-OCH3), 3.89 (s, 3H, 3-OCH3), 3.98 (s,
3H, 4′-OCH3), 3.99 (s, 3H, 7-OCH3), 6.37 (d, J = 2.1 Hz, 1H, H-6), 6.46 (d, J = 2.1Hz, 1H, H-
8), 7.00 (d, J = 8.4 Hz, 1H, H-5′), 7.70 (d, J = 1.8 Hz, 1H, H-2′), 7.75 (dd, J = 8.4, 2.1 Hz, 1H,
H-6′), 12.65 (s, 1H, 5-OH). 13C NMR (100 MHz, CDCl3, 25°C) δ (ppm): 178.75, 165.46,

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162.05, 156.73, 155.84, 151.39, 148.78, 138.99, 122.94, 122.18, 111.28, 110.86, 106.05,
97.83, 92.22, 60.20, 56.07, 56.00, 55.82.
The complete methylation of quercetin was accomplished only with a large excess of
iodomethane. Penta-O-methylquercetin was obtained after purification by flash
chromatography (EtOAc/hexane: 50-100%) as a white solid (yield: 52%, Rf = 0.92
o
1
(EtOAc), mp = 152-154 C). As for Retusin, the obtained product has the same H NMR
1
as described in the literature (Wang et al., 2005). H-NMR (400 MHz, CDCl3, 25°C) δ
(ppm): 3.89 (s, 3H, 3′-OCH3), 3.92 (s, 3H, 3-OCH3), 3.97 (b, 9H, 5-OCH3, 7-OCH3, 4′-OCH3),
6.36 (d, J = 2.1 Hz, 1H, H-6), 6.52 (d, J = 2.4 Hz, 1H, H-8), 6.99 (d, J = 9 Hz, 1H, H-5′), 7.70
(d, J = 1.8 Hz, 1H, H-2′), 7.73 (b, 1H, H-6′). 13C NMR (100 MHz, DMSOd6, 25°C) δ (ppm):
147.02, 163.88, 161.02, 158.79, 152.54, 150.80, 148.67, 141.19, 12341, 121.61, 111.27,
110.77, 109.49, 95.74, 92.43, 59.94, 56.41, 56.06, 55.96, 55.76.
Cell culture
Mouse MA-10 Leydig cells (Ascoli, 1981), provided by Dr. Mario Ascoli (University of
Iowa, Iowa City, Iowa), were grown in Dulbecco modified Eagle medium (DMEM)/F12
medium supplemented with 15% horse serum for transfections and viability assays. This
well-studied tumor Leydig cell line originated spontaneously in a C57Bl/6J mouse
(Ascoli, 1981) and retained many characteristics of their non-transformed counterparts.
For instance, MA-10 cells are able to produce progesterone in response to both
hormonal stimulations (LH and hCG) and cAMP analogues (Ascoli, 1981; Kilgore and
Stocco, 1989). Rat R2C Leydig cells were obtained from the ATCC (Manassas, VA, USA)
and were grown in DMEM/F12 medium supplemented with 2.5% fetal bovine serum
and 15% horse serum for viability assays. R2C tumor Leydig cells are known for their
constitutively high levels of steroidogenesis (Shin et al., 1968). Cells were cultured at
37°C and 5% CO2.
Cell viability
Following platting in 96 wells, treatments and incubations, the viability of MA-10 and
R2C treated cells was measured using crystal violet staining cell viability assay. Briefly,

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cultured cells were fixed using 10% formalin, stained with 0.25% crystal violet and
washed with distilled water. Retained coloration was then solubilised in 33% glacial
acetic acid and quantified by absorbance at 595 nm using a multimode microplate
reader (Varioskan, Thermo Scientific, Waltham, MA, USA).
Plasmids and transfections
The pCreb-luc and pNFkB-luc reporter plasmids constructs harboring 4 consensus
regulatory elements for Creb or NFkB, respectively, were purchased from Signosis Inc.
(Santa Clara, CA, USA). The murine -902 to +17 bp Star, -970 to +37 bp Cyp11a1, as well
as -1003 to +45 pb Fdx1 luciferase promoter constructs have been described previously
(Mendoza-Villarroel et al., 2014; Tremblay and Viger, 2001). MA-10 cells were
transfected using the jetPRIME® reagent according the manufacturer’s instructions
(Polyplus Transfection, Illkirch, France) followed by treatments using medium without
serum and analysis of lysates for luciferase activity as previously described (Martin et al.,
2009, 2008). Treatments were done using increasing concentrations (0, 10, 50, 250 µM)
of indicated flavonols for 6 h of incubation with or without 10 µM FSK (an activator of
the adenylate cyclase) in a total final concentration of 1.75% DMSO. Stimulations for 6h
with 10 µM FSK have been used based on their capacity to increase steroidogenic genes
expression within Leydig cells (Martin et al., 2008). Concentrations of flavonols (10-250
µM) used in this study are based on preliminary data and previously published literature
(Y.-C. Chen et al., 2007).
Quantitative real-time PCR
Following treatments, RNA was isolated from cultured MA-10 Leydig cells using the
E.Z.N.A. Total RNA kit (Omega Bio-Tek, Inc., Norcross, GA). The purity and concentration
of the isolated RNA was assessed using a Nanodrop 1000 where 260/280 ratios were
approximately 2.1. The qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD)
was used to synthesize cDNA. Quantitative real-time polymerase chain reaction (qPCR)
was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad,
Hercules, CA) on a CFX Connect™ Real-Time PCR Detection System (Bio-Rad). The

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primers used for Star, Cyp11a1 and Rpl19 quantifications have been described
previously (Cormier et al., 2017), whereas Creb1 expression was evaluated using
forward (5’- CAG AGT GGA GAT GCT GCT GT-3’) and reverse (5’-GAT GTT GCA TGA GCT
GCT GG-3’) oligos. Primers specificities were assessed by melt curve analysis and gel
electrophoresis of DNA products. Gene expression levels were evaluated by the ΔΔ
threshold cycle (Ct) method after confirmation that amplification efficiency was
between 90% and 110% for all primer pairs. The reference gene Rpl19 mRNA was used
as control for total mRNA recovery. Experiments were repeated four times and
quantified as technical duplicates.
Progesterone quantification
ELISAs for progesterone quantification were performed as recommended by the
manufacturer (Oxford Biomedical Research, Rochester Hills, MI). Following treatments,
the culture medium was transferred to 1.5-mL tubes and stored at -80°C until ELISAs
were performed. For basal progesterone quantification (without FSK stimulation), 50 µL
of medium was used undiluted in the ELISA. For FSK-stimulated cells, 10 µL of the
medium was diluted with 90 µL EIA buffer (1:10), 50 µL of which was subsequently used
in the ELISA.
Statistical analyses
To identify significant differences between groups, statistical analyses were done on fold
activation results using a two-way analysis of variance for polyphenolics concentration
and presence or absence of FSK, followed by the Tukey’s multiple comparison test. For
all statistical analyses, P < 0.05 was considered significant. All statistical analyses were
done using the GraphPad Prism 6.07 software package (GraphPad Software Inc, La Jolla,
CA).

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Results
Flavonols and quercetin derived compounds do not decrease viability of
Leydig cell lines
To ensure that flavonols and quercetin derivative compounds used in this study have no
negative effects on Leydig cells’ viability, we incubated two Leydig cell lines models with
increasing doses of these compounds in absence or presence of the activator of
adenylate cyclase FSK for 6h (Fig. 2). For mouse MA-10 Leydig cells, increasing levels of
quercetin resulted in increased viability by more than 1.8 folds (Fig. 2a), whereas other
polyphenolics had no significant effects compared to unstimulated cells (Fig. 2b-e). For
rat R2C Leydig cells, myricetin at 250 µM stimulated cell viability by more than 1.7 folds
(Fig. 2g), whereas stimulations with other compounds had no influence on viability
compared to unstimulated cells (Fig. 2f, h-j). Thus, synthetic compounds derived from
quercetin (pentaacetylquercetin, retusin and penta-O-methylquercetin) had no negative
effects on Leydig cells’ viability under the conditions investigated.
Quercetin and its derivatives increase cAMP-dependent Creb activation in MA-
10 Leydig cells
It is well established that the cAMP-response element binding protein (Creb) is an
important activator of the expression of steroidogenic genes in Leydig cells. Thus,
changes in its activity are strong indicators of the state of the cAMP/PKA pathway, as
well as testosterone production. Here we looked at the influence of flavonols and
quercetin derived compounds on Creb transcriptional activity by transfection of a
reporter construct harboring 4 consensus regulatory elements for Creb in MA-10 Leydig
cells, followed by stimulations with increasing concentrations of indicated polyphenolics
for 6 h with or without FSK (Fig. 3). Although none of the polyphenolics investigated
had significant effect alone, stimulations with 10 µM of quercetin, pentaacetylquercetin
or retusin further increased FSK-dependent stimulation of Creb transcriptional activity
(Fig. 3a, c, d). FSK-dependent stimulatory effect of Creb activity by penta-O-
methylquercetin required treatments with higher concentrations (50-250 µM) (Fig. 3e).
Interestingly, FSK-dependent increase in Creb transcriptional activity was only

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maintained in the presence of high levels of retusin and penta-O-methylquercetin (Fig.
3d, e).
To investigate if the effects of flavonols and quercetin derived compounds were specific
to genes important for steroidogenesis in Leydig cells, stimulations were also done after
transfection of a reporter construct harboring 4 consensus regulatory elements for NFkB
in MA-10 cells. As shown in Fig. 4, high levels of quercetin and pentaacetylquercetin
(250 µM) resulted in repression of the activity of NFkB transcription factors. Under all
conditions investigated, NFkB transcriptional activity was independent of adenylate
cyclase activation using 10 µM FSK. However, addition of 10 µM quercetin increased
NFkB transcriptional activity in response to FSK by more than 1.5 fold (Fig. 4a). NFkB
transcriptional activity was also increased in response to treatment of Leydig cells with
increasing concentrations of retusin (Fig. 4d).
Flavonols have dose-dependent stimulatory effects on cAMP-dependent
activations of steroidogenic genes promoters
Next, we wanted to confirm if stimulations of Creb transcriptional activity by flavonols
and quercetin derived compounds had similar consequences on steroidogenic
promoters’ activations. MA-10 Leydig cells were transfected with a murine -902 to +17
bp Star promoter reporter construct, followed by stimulations with increasing
concentrations of polyphenolics. Interestingly, myricetin (10 µM) increased FSK-
dependent activation of the Star promoter within MA-10 Leydig cells (Fig. 5b). For all the
flavonols and quercetin derived compounds tested, use of 250 µM treatments resulted
in complete loss of FSK-dependent increase in Star promoter activity (Fig. 5).
Following entry of cholesterol inside mitochondria of Leydig cells, it is then metabolised
to pregnenolone by the steroidogenic enzyme Cyp11a1. Using a -970 to +37 bp Cyp11a1
promoter reporter construct, we looked at the influences of flavonols and quercetin
derived compounds on its transcriptional regulation (Fig. 6). As for Star, high levels of
the investigated compounds had no stimulatory effect on FSK-dependent regulation of
Cyp11a1 promoter activity within MA-10 Leydig cells. However, use low to intermediate

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levels of quercetin and pentaacetylquercetin (10-50 µM) resulted in increased FSK-
dependent activation of the Cyp11a1 promoter (Fig. 6a, c). Moreover, quercetin alone
(10-50 µM) was sufficient to upregulate Cyp11a1 promoter activity within MA-10 Leydig
cells (Fig. 6a).
Supporting steroid biosynthesis through electron transfer to Cyp11a1, transcriptional
regulation of Fdx1 expression by flavonols and quercetin derived compounds was also
investigated by transfecting MA-10 Leydig cells with a mouse -1003 to +45 pb Fdx1
promoter reporter construct. As shown in Fig. 7, only quercetin (10 µM) and retusin (50
and 250 µM) improved FSK-dependent stimulation of Fdx1 promoter activity (Fig. 7a, d).
High levels of quercetin, myricetin and pentaacetylquercetin inhibited FSK-induced Fdx1
promoter activation (Fig. 7a-c), further contributing to reduced Cyp11a1 enzymatic
activity under these conditions. Interestingly, 10 µM of pentaacetylquercetin was the
only treatment able to increase basal Fdx1 promoter activity within MA-10 Leydig cells
(Fig. 7c). Although cAMP-dependent activation of the Fxd1 promoter was preserved in
the presence of 250 µM penta-O-methylquercetin, activities were lower than basal and
FSK-induced Fdx1 promoter regulations (Fig. 7e).
Results for pentaacetylquercetin (10 µM) using luciferase reporter assays were
confirmed at the mRNA level using qPCR quantification. Although pentaactetylquercetin
(10 µM) had no effect on Creb1 mRNA expression (Fig. 8a), this quercetin derivative
increased FSK-dependent activation of Star transcription from MA-10 Leydig cells (Fig.
8b). In addition, pentaacetylquercetin tended to improve FSK-dependent activation of
Cyp11a1 expression from MA-10 cells, however such effect was non-significant
compared to FSK alone (Fig. 8c).
Pentaacetylquercetin increases cAMP-dependent progesterone production
from MA-10 Leydig cells
To confirm our results regarding the influences of flavonols and quercetin derived
compounds on transcriptional regulation of steroidogenic genes within Leydig cells, we
quantified progesterone accumulation within the cell culture medium following 6h of

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stimulations using the indicated conditions (Fig. 9). As expected, treatments with 10 µM
FSK resulted in more than 14 fold increase in progesterone accumulation. Only
pentaacetylquercetin 10 µM had a stimulatory effect on cAMP-dependent accumulation
of progesterone from MA-10 Leydig cells, further increasing its concentration by 3 folds
compared to treatment with FSK only. Surprisingly, treatments with other compounds
had no effect on basal and cAMP-dependent accumulation of progesterone from MA-10
cells. However, FSK-dependent increases of progesterone accumulation in response to
retusin and penta-O-methylquercetin were not significant.

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Discussion
In the current study, we show that flavonols and quercetin derived compounds do not
decrease viability of mouse MA-10 and rat R2C Leydig cell lines. Interestingly, higher
concentrations of quercetin resulted in increased MA-10 Leydig cells’ viability. However,
such increased viability by quercetin didn’t translate into improved progesterone
production. We also demonstrate that low levels of flavonols and quercetin derived
compounds stimulate cAMP-dependent, whereas high levels of these compounds
downregulate basal and cAMP-dependent, steroidogenic genes expression within MA-
10 Leydig cells. Interestingly, pentaacetylquercetin was able to increase cAMP-
dependent progesterone synthesis.
Quercetin improves Creb transcriptional activity, as well as Cyp11a1 and Fdx1
promoters’ activities
Known to be an important activator of steroidogenic genes expression in Leydig cells
(King and LaVoie, 2012; Manna et al., 2002), Creb1 stimulates Star expression in Leydig
cells (Manna et al., 2009, 2003). Low levels of quercetin, as well as pentaacetylquercetin
or retusin improved FSK-dependent stimulation of Creb transcriptional activity, whereas
higher concentrations of penta-O-methylquercetin was required to generate a similar
effect. FSK-dependent increase in Creb transcriptional activity was only maintained by
high levels of retusin. In addition to Creb, quercetin also improved cAMP-dependent
stimulation of NFkB transcriptional activity. Since NFkB target genes involve anti-
apoptotic proteins and cell cycle regulators that promote cancer cells proliferation
(Karin and Lin 2002), such dysregulation of NFkB by quercetin, may have contributed to
increased MA-10 Leydig cells viability. As shown previously with stimulations of Leydig
cells with flavones (unpublished data), high levels of quercetin contributed to decrease
Creb1 and NFkB transcriptional activities, as well as cAMP-dependent Star and Fdx1
promoters’ activations. Others have shown that quercetin had no adverse effects on
Leydig cells for concentrations up to 60 µM being investigated (Hu et al., 2015). Such

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discrepancy may be attributed to differences in cell culture conditions as well as the use
of rat primary Leydig cells compared to MA-10 immortalized Leydig cells.
Although quercetin non-significantly improved cAMP-dependent Star promoter
activation under our experimental conditions, others have reported that quercetin
increased Star mRNA levels, Star promoter activity and steroid hormone production
from MA-10 Leydig cells (Y.-C. Chen et al., 2007). Quercetin reduced Dax1 (dosage-
sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1)
protein content within Leydig cells, contributing to further increase Star protein
synthesis in response to cAMP stimulation (Wang, 2011). Quercetin, as well as other
flavonoids such as chrysin, apigenin and luteolin, have also been shown to enhance Star
gene expression and steroidogenesis in Leydig cells by blocking the cyclooxygenase-2
(Cox2)-dependent signaling. In aging males, increased Cox2 expression in Leydig cells
results in decreased Star gene expression and testosterone production (Wang et al.,
2005). Reduction of Cox2 by quercetin is due to inhibitory effects on several
transcription factors, such as NFkB (C.-Y. Chen et al., 2007; Ha et al., 2010; Liang et al.,
1999), an important regulator of Cox2 transcription. Indeed, quercetin reduced NFkB
activity by inhibiting IKK/IkB signaling, leading to reduced NFkB nuclear translocation
(García-Mediavilla et al., 2007). In our stimulation conditions, inhibitory effects of
quercetin on NFkB transcriptional activity are only observed using high concentrations
and would require further investigation for its physiological relevance in vivo. In addition
to NFkB signaling, quercetin may also inhibit PI3K activity and Akt phosphorylation,
resulting in downregulation of Cox2 expression in Leydig cells, as shown previously in
liver epithelial cells (Lee et al., 2010). Members of the NFkB family are also known to
cooperate with CCAAT/enhancer binding protein beta (Cebpb) to upregulate Nr4a1
(Nur77) expression, leading to increased steroidogenesis within MA-10 Leydig cells (El-
Asmar et al., 2009). Thus, combination of low levels of quercetin with FSK may increase
NFkB transcriptional activity and participate in a dose dependent regulation of
steroidogenic promoters through Cebpb and Nr4a1 within MA-10 Leydig cells.

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Although low levels of quercetin increased basal and FSK-stimulated Cyp11a1 as well as
Fdx1 promoters’ activities, such effect didn’t translate into increased progesterone
synthesis by MA-10 Leydig cells. Interestingly, quercetin alone (10-50 µM) was sufficient
to upregulate Cyp11a1 promoter activity. However, others have shown that stimulation
of Leydig cells isolated from 28 days old Wistar rats with quercetin 50 µM for 6h add no
effect on steroidogenic genes expression (Abarikwu et al., 2013). Moreover, daily
administration of 50 mg/kg of quercetin to adult male Sprague-Dawley rats for 70 days
had no effect on plasma testosterone concentration (Saber et al., 2016). Quercetin may
rather be important to suppress oxidative stress, as shown previously in rat Leydig cells
(Hu et al., 2015). Such regulation may involve the antioxidant transcription factor Nrf2,
known to protect against oxidative stress by nuclear translocation and association to
ARE regulatory elements. Thus, quercetin supplementation could prevent the reduction
in testosterone production by adult Leydig cells, as oxidative stress is mainly responsible
for such decline in aging males (Cao et al., 2004; Chen et al., 2015).
Myricetin increases cAMP-dependent Star promoter activation
Although myricetin did not significantly improved FSK-dependent stimulation of Creb
transcriptional activity in MA-10 Leydig cells, this flavonol had a positive influence on
Star promoter activation resulting in an increase of progesterone production by 2 folds
(though non-significant). As quercetin, high levels of myricetin also inhibited cAMP-
dependent increase in Creb1 transcriptional activity, as well as Star and Fdx1 promoters’
activations. Other flavonoids such as chrysin and apigenin have been shown to increase
steroid hormone production from MA-10 Leydig cells and such increase also required
Star activation (Jana et al., 2008; Li et al., 2011). Within Leydig cells, these compounds
have been suggested to upregulate Star expression through inhibition of the
transcription factor Dax1 (Jana et al., 2008), an important repressor of steroidogenic
genes expression. However, the implication of this regulatory mechanism in the
regulation of cAMP-dependent Star expression by myricetin remains to be confirmed.

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Sub-threshold levels of cAMP stimulation (0.05 and 0.1 mM) have been used to enhance
Leydig cells’ steroidogenesis in response to flavones (Jana et al., 2008; Li et al., 2011).
These concentrations are usually unable to induce significant increases in steroid
hormone and Star protein expression in MA-10 cells (Wang et al., 2008). Even though
FSK increased endogenous cAMP production by adenylate cyclase under our stimulation
condition (Stocco and Chaudhary, 1990), such increase may not be sufficient to promote
Star expression by flavonols and/or quercetin derived compounds within MA-10 Leydig
cells. How myricetin is able to increase cAMP-dependent stimulation of Star expression
within Leydig cells is unknown and needs further investigation.
Pentaacetylquercetin improves progesterone synthesis by Leydig cells
Addition of five acetyl groups to quercetin had no major effect on the capacity of
quercetin to activate Creb1 in response to FSK. However, such modification resulted in
complete loss of cAMP-dependent increase of Fdx1 promoter activation. Interestingly,
having a positive improvement effect on Cyp11a1 steroidogenic promoter activation
and Star mRNA expression by cAMP, pentaacetylquercetin led to increased
progesterone accumulation from MA-10 Leydig cells in response to FSK. However, such
increase cannot be explained by modulation of other steroidogenic genes expressions
and may involve enhanced steroidogenic proteins activities through post-translational
modifications (phosphorylation) and/or inhibition of steroids degradation.
Interestingly, high levels of pentaacetylquercetin had similar inhibitory effects as
quercetin: inhibiting Creb1 and NFkB transcriptional activities, as well as Star, Cyp11a1
and Fdx1 steroidogenic promoters’ activations. Others have also showed that
pentaacetylquercetin inhibited COX2 expression in RAW 264.7 cells (Chen et al., 2001).
As for quercetin, this may result into increased Star expression by pentaacetylquercetin
(although non-significant under our stimulation conditions) in response to co-
treatments with FSK of MA-10 Leydig cells.
In addition to modulation of the cAMP/PKA pathway, flavones, such as apigenin and
luteolin, are also able to inhibit the activation of the mitogen-activated protein kinases

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(MAPK) (Hwang et al., 2011), being essential for activation of several transcription
factors, such as Creb, Cebp and NFkB, normally involved in upregulation of Cox2
expression in different tissues (Choi and Lee, 2010; Chun and Surh, 2004; Tsatsanis et
al., 2006). Indeed, inhibitory effects of apigenin on MAPK activation reduces Cox2
transcription in breast cell lines (Yi Lau and Leung, 2010) and may also be involved in
cAMP-dependent increase in Star promoter activity by myricetin. Pentaacetylquercetin
may also modulate the MAPK pathway to increase Creb1 transcriptional activity, as well
as Star and Cyp11a1 expressions in Leydig cells, contributing to increased progesterone
production. Indeed, numerous other flavonoids such as butein, genistein, paeonol, 3’-
hydroxyflavone and curcumin have been shown to modulate the activity of the MAPK
pathway in other cell types (Niu et al., 2015).
Retusin increases cAMP-dependent Fdx1 promoter activation
Treatments with increasing concentrations of retusin resulted in improved NFkB
transcriptional activity within Leydig cells. In addition to quercetin, retusin also
improved FSK-dependent stimulation of Fdx1 promoter activity. However, such
stimulatory effect from retusin was not sufficient to improve progesterone production
in response to stimulation of the cAMP/PKA pathway. Interestingly, Creb1 and NFkB
transcriptional activities, as well as steroidogenic promoters’ activations were
maintained in the presence of high levels of retusin. Thus, the presence of four
hydroxymethyl groups on quercetin may reduce its cytotoxicity and may be better
tolerated at high concentrations in vivo.
Compared to retusin, only Creb1 transcriptional activity could be maintained in the
presence of high levels of penta-O-methylquercetin, suggesting that addition of a
hydroxymethyl group in position 5 may have a detrimental effect on transcriptional
regulation by this compound in Leydig cells. Although cAMP-dependent activation of the
Fxd1 promoter was preserved in the presence of high levels of penta-O-
methylquercetin, activities were lower than basal and FSK-induced Fdx1 promoter
regulations.

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As shown with chrysin, testosterone concentration may also be increased by preventing
its conversion into estradiol through inhibition of aromatase activity (Dhawan et al.,
2002). Thus, even though flavonols and quercetin derived compounds other than
pentaacetylquercetin do not promote progesterone accumulation from MA-10 Leydig
cells in our stimulation conditions, they may contribute to increased testosterone
accumulation as a result of decreased degradation. Indeed, quercetin has been shown
to inhibit aromatase activity in other cell models (Park et al., 2015).
Current finding regarding flavonols and quercetin derived compounds on Leydig cells’
steroidogenic genes regulation remain to be confirmed using animal studies and are
promising for improvement of endocrine and reproductive functions. Indeed, potential
applications of polyphenolics in delaying the decline of testosterone in aging men have
been supported by animal studies on flavonoid-enhanced testosterone production and
reproductive function. Supplementation with chrysin improved reproductive function of
2-year-old male rats (Dhawan et al., 2002). Lifetime exposure to flavonoids, such as
daidzein, glycitein and genistein, also increased serum and testicular testosterone levels
in rats (McVey et al., 2004). In addition, injections with catechins or its derivatives also
increased testosterone plasma concentrations (Yu et al., 2010). Beneficial effects of
quercetin were reported in diabetic rats, where sperm viability and motility was
improved, possibly as a result of increased serum total testosterone (Khaki et al., 2010).
Most flavonoids are rapidly metabolized within the intestinal mucosa and liver, making
the bioavailability of flavonoids and their metabolites generally low (Manach et al.,
2005). Indeed, plasma concentrations of flavonoids and those to which the Leydig cells
are exposed are in the low micromolar range. Thus, concentrations of 250 µM of a
particular flavonoid would be difficult to attain with a normal diet and may only be
relevant with the consumption of specific supplements. Use of natural or synthetic
flavonoids to improve the health of aging males will require long-term animal studies
and clinical studies.

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In conclusion, pentaacetylquercetin effectively increased cAMP-dependent
accumulation of progesterone from MA-10 Leydig cells, possibly through activation of
Star and Cyp11a1 transcriptions. Thus, dietary supplementation of pentaacetylquercetin
may be effective to maintain testosterone production within aging males. However,
although there is a correlation between increased Star and Cyp11a1 expressions and
stimulation of steroidogenesis within testicular Leydig cells, more research will be
required to clearly define the regulatory mechanism of pentaacetylquercetin on
testosterone synthesis.

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Acknowledgements
Current work was funded by the New Brunswick Innovation Foundation (NBIF)
(#IAR2015 to L.J.M.; #RIF2015-014 to M.T.) and the Natural Sciences and Engineering
Research Council (NSERC) of Canada (#386557 to L.J.M.; #04560 to M.T.).
Disclosure
The authors declare that there is no conflict of interest that would prejudice there
impartiality.

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Figure legends
Figure 1. Chemical structures of the flavonols quercetin and myricetin, as well as their
derivatives pentaacetylquercetin, retusin and penta-O-methylquercetin.
Figure 2. Viability of MA-10 Leydig cells in response to treatments with flavonols and
quercetin derived compounds. Exponentially growing cells were incubated in the
absence or presence of increasing doses of indicated flavonols or quercetin derived
compounds (10, 50 or 250 µM) for 6h with or without the adenylate cyclase activator
FSK at 10 µM, followed by determination of cell viability as described in Material and
methods. The results are presented as fold viability over control (DMSO only) (± SEM).
Comparisons to control were done using a one-way ANOVA followed by Tukey’s
multiple comparison test. A different letter denotes a significant difference (P<0.05).
Figure 3. Effects of increasing levels of A) quercetin, B) myricetin, C)
pentaacetylquercetin, D) retusin and E) penta-O-methylquercetin on pCREB-luc activity.
MA-10 Leydig cells were transfected with a luciferase plasmid reporter harboring 4
consensus CREB regulatory elements upstream of the gene for firefly luciferase. Cells
were treated with increasing doses of indicated flavonols or quercetin derived
compounds (10, 50 or 250 µM) for 6h of incubation with or without the adenylate
cyclase activator FSK at 10 µM. Results are shown as Fold Activation over control (DMSO
only) (± SEM). A two-way ANOVA was used to analyze data according to concentrations
of polyphenolics and stimulation with FSK, followed by Tukey’s multiple comparison
test. A different letter denotes a significant difference (P<0.05).
Figure 4. Effects of increasing levels of A) quercetin, B) myricetin, C)
pentaacetylquercetin, D) retusin and E) penta-O-methylquercetin on pNFκB-luc activity.
MA-10 Leydig cells were transfected with a luciferase plasmid reporter harboring 4
consensus NFkB regulatory elements upstream of the gene for firefly luciferase. Cells
were treated with increasing doses of indicated flavonols or quercetin derived
compounds (10, 50 or 250 µM) for 6h of incubation with or without the adenylate
cyclase activator FSK at 10 µM. Results are shown as Fold Activation over control (DMSO

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only) (± SEM). A two-way ANOVA was used to analyze data according to concentrations
of polyphenolics and stimulation with FSK, followed by Tukey’s multiple comparison
test. A different letter denotes a significant difference (P<0.05).
Figure 5. Effects of increasing levels of A) quercetin, B) myricetin, C)
pentaacetylquercetin, D) retusin and E) penta-O-methylquercetin on mouse Star
promoter activity. MA-10 Leydig cells were transfected with a luciferase plasmid
reporter harboring the -902 to +17 bp mouse Star promoter upstream of the gene for
firefly luciferase. Cells were treated with increasing doses of indicated flavonols or
quercetin derived compounds (10, 50 or 250 µM) for 6h of incubation with or without
the adenylate cyclase activator FSK at 10 µM. Results are shown as Fold Activation over
control (DMSO only) (± SEM). A two-way ANOVA was used to analyze data according to
concentrations of polyphenolics and stimulation with FSK, followed by Tukey’s multiple
comparison test. A different letter denotes a significant difference (P<0.05).
Figure 6. Effects of increasing levels of A) quercetin, B) myricetin, C)
pentaacetylquercetin, D) retusin and E) penta-O-methylquercetin on mouse Cyp11a1
promoter activity. MA-10 Leydig cells were transfected with a luciferase plasmid
reporter harboring the -970 to +37 bp mouse Cyp11a1 promoter upstream of the gene
for firefly luciferase. Cells were treated with increasing doses of indicated flavonols or
quercetin derived compounds (10, 50 or 250 µM) for 6h of incubation with or without
the adenylate cyclase activator FSK at 10 µM. Results are shown as Fold Activation over
control (DMSO only) (± SEM). A two-way ANOVA was used to analyze data according to
concentrations of polyphenolics and stimulation with FSK, followed by Tukey’s multiple
comparison test. A different letter denotes a significant difference (P<0.05).
Figure 7. Effects of increasing levels of A) quercetin, B) myricetin, C)
pentaacetylquercetin, D) retusin and E) penta-O-methylquercetin on mouse Fdx1
promoter activity. MA-10 Leydig cells were transfected with a luciferase plasmid
reporter harboring the -1003 to +45 bp mouse Fdx1 promoter upstream of the gene for
firefly luciferase. Cells were treated with increasing doses of indicated flavonols

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quercetin derived compounds (10, 50 or 250 µM) for 6h of incubation with or without
the adenylate cyclase activator FSK at 10 µM. Results are shown as Fold Activation over
control (DMSO only) (± SEM). A two-way ANOVA was used to analyze data according to
concentrations of polyphenolics and stimulation with FSK, followed by Tukey’s multiple
comparison test. A different letter denotes a significant difference (P<0.05).
Figure 8. Effects of pentaacetylquercetin on FSK-dependent expression of steroidogenic
genes from MA-10 Leydig cells. For (A-C), MA-10 Leydig cells were stimulated with
pentaacetylquercetin (10 µM), in the absence or presence of FSK (10 µM) for 6h,
followed by mRNA extractions and quantitative real-time PCR assays. Results were
normalized using Rpl19 reference gene expression and reported to control (DMSO only).
The number of experiments, each quantified as technical duplicates, is indicated. Results
are shown as Fold expression (A-C) over control (DMSO only) (± SEM). A two-way
ANOVA was used to analyze data according to pentaacetylquercetin and FSK
treatments, followed by Tukey’s multiple comparison test. A different letter denotes a
significant difference (P<0.05).
Figure 9. Influences of flavonols and quercetin derived compounds on cyclic-AMP
dependent stimulation of progesterone production from MA-10 Leydig cells. MA-10
cells were treated with indicated polyphenolics at 10 µM with or without 10 µM FSK for
6 h, followed by cell culture medium progesterone quantification by EIA assay (ng/mL ±
SEM). A two-way ANOVA was used to analyze data according to stimulations with
polyphenolics and/or FSK, followed by Fisher’s LSD multiple comparison test. A different
letter denotes a significant difference (P<0.05).

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Figure 1